Abstract

In contrast to naïve T cells that recognize short antigen-derived peptides displayed by specialized antigen-presenting cells, immunoglobulin receptors of B lymphocytes primarily recognize intact proteins. How and where within a lymph node such unprocessed antigens become available for naïve B cell recognition is not clear. We used two-photon intravital imaging to show that, after exiting high-endothelial venules and before entry into lymph node follicles, B cells survey locally concentrated dendritic cells. Engagement of the B cell receptor by the dendritic cell (DC)–associated antigen leads to lymphocyte calcium signaling, migration arrest, antigen acquisition, and extrafollicular accumulation. These findings suggest a possible role for antigen-specific B-DC interactions in promoting T cell–dependent antibody responses in vivo.

Adaptive immunity depends on the activation of rare, antigen (Ag)-specific lymphocytes, which recirculate through blood and secondary lymphoid organs such as lymph nodes (LNs) in search of their Ags. T cell stimulation typically involves dendritic cell (DC) presentation of protein Ag fragments bound to major histocompatibility complex (MHC)–encoded molecules (1). In contrast, naïve B cells are thought to respond to intact free Ags that exit the lymph or the blood and enter the LN follicular area, within which most B cells reside. However, the lymph carrying Ags from peripheral tissues to the LN is channeled through sinuses and conduits that lie outside of the follicles and whose structure limits direct access of high molecular weight or particulate material to the LN parenchyma (2–4).

Given these constraints on antigen access, we considered the possibility that B cell activation may also use a system of active cell-aided Ag delivery. Consistent with this hypothesis, injection of DCs carrying protein, bacterial, or viral Ags leads to the induction of specific antibodies (5–9). Furthermore, DCs that capture bacteria from the circulation have been shown to interact with and promote activation of splenic marginal zone B cells (10), and DCs are known to maintain intracellular pools of undegraded Ags that can be recycled to the cell surface (5, 11–13). To test our hypothesis directly, we used the well-studied protein antigen hen egg lysozyme (HEL), which is highly cationic and taken up efficiently into cells through electrostatic interactions with anionic surface molecules, thus simulating normal receptor-mediated Ag uptake by DCs. B cells from HEL-specific B cell receptor (BCR) transgenic MD4 mice (14) showed rapid elevation of intracellular calcium concentration ([Ca2+]) upon coculture with HEL-pulsed DCs (HEL-DCs) (Fig. 1A) followed by increased surface expression of CD86 and MHC class II molecules (Fig. 1B). Such activation was abrogated if the B cells and DCs were separated by a cell-impermeable membrane (fig. S1), making it unlikely that MD4 B cells had simply been triggered by Ags released from viable or dead DCs. To examine this issue further, we pulsed DCs with HEL at 37°C or on ice and then treated them with pronase to remove surface-associated HEL. Although DCs pulsed at 37°C had a lower amount of surface-associated HEL than those pulsed on ice, they nevertheless acquired a much larger total amount of Ags (Fig. 1C, left), indicating that HEL acquired by DCs at physiological temperature is predominantly intracellular. When pronase was used to remove HEL from the DC surface (Fig. 1C, right), DCs pulsed at 37°C retained the ability to activate MD4 B cells, whereas those pulsed on ice did not (Fig. 1D); again, membrane separation of the cells prevented the response (fig. S1). Thus, HEL endocytosed by DCs can reach the cell surface in a form able to activate B cells in vitro.

B cell activation by Ag-carrying DCs in vitro and in LNs. (A and B) Naïve MD4 or control non-tg B cells were mixed with HEL-pulsed DCs or unpulsed DCs or left in medium alone and then immediately assayed for intracellular Ca2+ or maintained in culture for 12 hours before being tested for surface expression of CD86 and I-Ab. (A) Fluo-4 (20) fluorescence normalized against the forward scatter (FSC, an indicator of the cell volume) was used to gauge the relative intracellular [Ca2+]. (B) CD86 and I-Ab expression on CD19+ cells from coculture of the indicated B-DC combinations. (C) DCs pulsed under the indicated conditions were washed and assayed for surface-associated or total-cell-associated HEL by antibody staining before or immediately after being treated with pronase. (D) Pronase-treated or sham-treated DCs were maintained in culture for 20 hours before being cocultured with MD4 B cells for an additional 12 hours. CD86 expression was then assessed on CD19+ B cells. All results are representative of at least three independent experiments. (E and F) Intravital observation of B cell calcium responses induced by Ag-bearing DCs in LNs. (E) Time-lapse images of the intracellular Ca2+ response, revealed by Fluo-4 (green), of an MD4 cell (red) upon contact with an HEL-DC (20) (movieS1). Scale bar represents 10 μm. (F) Time-lapse images of an MD4 cell that exited an HEV, came into contact with an HEL-DC, and responded with an elevation of intracellular Ca2+. Dotted lines approximate the HEV border. (Insets) The B cell border (without the cytoplasmic stain) for better differentiation of the B cell-associated Fluo-4 fluorescence from that of the HEV labeled with fluorescein isothiocyanate (FITC)–dextran (movie S2). Scale bar, 10 μm.

Although these data demonstrate that B cell activation can be induced directly by Ag-carrying DCs in culture, in LNs the majority of DCs reside in the T cell zone (15), raising the question of how naïve B cells might access DC-associated Ags in vivo. The region surrounding the high-endothelial venules (HEVs) is potentially an ideal place for B cells to scan DC-associated Ags; because this is the first parenchymal location of naïve B cells on their way to the follicle, it is a region rich in migrating Ag-laden DCs (16, 17), and it is also where resident DCs sample Ags arriving via the LN conduits (18). Consistent with this idea, LN sections showed that recent immigrant B cells initially concentrated around HEVs before entering follicles (figs. S2 and S3), colocalizing with and physically contacting DCs (fig. S4).

Next, intravital two-photon microscopy (19) was used to determine whether these apparent DC-B interactions around HEVs result in B cell activation by DC-associated Ag. To look for an early sign of Ag stimulation, we examined Ca2+ responses of MD4 B cells in the LNs of living mice that had been transferred with HEL-DCs or control ovalbumin (OVA)-pulsed DCs (20). Upon contact with dendrites extending from HEL-DCs, MD4 B cells exhibited an abrupt increase in intracellular [Ca2+], often followed by signal oscillations during an extended period of association (Fig. 1E and movie S1). Coincident with this Ca2+ response, B cells typically lost their initial polarized shape, similar to what has been observed with Ag-triggered T lymphocytes (21, 22). Of 159 MD4 cells visualized contacting HEL-DCs, 45 showed detectable elevation of intracellular [Ca2+], a frequency five times higher than that seen when MD4 cells contacted OVA-DCs (8 out of 143; P < 0.01). By using covisualization of HEVs, we also documented such Ca2+ responses in MD4 cells that exited HEVs and immediately engaged HEL-DCs (Fig. 1F and movie S2). Together, these data indicate that, upon contact with Ag-bearing DCs, receptor-dependent signaling can be triggered in LN B cells shortly after their exit from HEVs and before migration into the follicle.

T cells show prolonged interactions with DCs upon recognition of peptide-MHC ligands in vivo (23, 24), and elevation of intracellular [Ca2+] is linked to reduced migration by receptor-engaged T cells and thymocytes (22). We therefore undertook a detailed survey in which the migration behavior of each B cell was documented from the moment of its entry into a LN through an HEV. This analysis revealed that MD4 B cells bound to HEV-proximate HEL-DCs (Fig. 2A and movie S3) for considerably longer periods than they bound to OVA-DCs (Fig. 2A and movie S4). In contrast, immigrating nontransgenic (non-tg) B cells made only brief contact with either HEL- or OVA-DCs, consistent with a lack of specific Ag recognition (movies S5 and S6). Initial MD4 B cell associations with HEL-DCs lasted three times as long as those involving Ag-nonspecific B-DC combinations (P = 0.0004) (Fig. 2B). There was also a reduction in the migration speed of MD4 cells interacting with HEL-DCs compared with OVA-DCs or non-tg B cells interacting with either HEL- or OVA-DCs (Fig. 2B). MD4 cells that had not interacted with any transferred HEL-DCs moved more rapidly than those that had physically engaged Ag-bearing DCs (median instantaneous velocities of 4.8 versus 3.4 μm/min from 405 versus 882 measurements, P = 3 × 10–17). The velocities of the MD4 cells failing to contact an HEL-DC were comparable to those of non-tg B cells that failed to contact transferred DCs (median instantaneous velocities of 4.8 versus 4.8 μm/min from 405 versus 658 measurements, P = 0.7), although the velocities of both B cell types were lower than their respective migration speeds within the follicles of nonimmunized mice, presumably reflecting Ag-nonspecific inflammatory and/or activated DC-related factors influencing B cell motility (fig. S5). Thus, in a LN containing HEL-DCs, immigrating MD4 B cells only showed appreciable motility changes if they directly contacted the Ag-carrying DCs. This implies that DCs released little or no free Ag into the extracellular space of the LN, at least in a form or quantity able to evoke a measurable change in B cell mobility, consistent with the result from the in vitro transwell assays (fig. S1).

Contact-dependent, Ag-specific interactions between recent immigrant B cells and Ag-carrying DCs in the peri-HEV region of LNs. (A) Timelapse images of DC interactions with MD4 B cells as the latter exit HEVs. “DC” in white denotes a DC in contact with an MD4 B cell (circle), whereas “DC” in red indicates a DC from which an MD4 cell has disengaged. (B) (Left) Scatter plots of the duration of individual DC contacts made by B cells imaged as they leave HEVs. The lines indicate median values. (Right) The median instantaneous velocity of B cells leaving HEV during and after contacts with DCs. N is the number of velocity measurements in each category. Data were compiled from more than 30 imaging sessions that captured 33 MD4 B cells as they exited HEV and interacted with DCs (23 with HEL-DCs and 10 with OVA-DCs) and that also captured 20 non-tg B cells imaged under the same conditions (eight with HEL-DCs and 12 with OVA-DCs). (C) From these same data sets, typical migration patterns of recent immigrant B cells within and around DC arrays (blue) in the peri-HEV region were examined. Blood vessels are in green. HEV segments were identified on the basis of intravascular attachment of transferred B cells. Migration tracks are highlighted in red, with the actual B cell fluorescence omitted for clarity (movie S7). (D) Contact time (left), average cell migration speed (center), and distribution of instantaneous velocities (right) of MD4 and non-tg B cells cotransferred into HEL-DC or OVA-DC recipients. Data were compiled from four independent experiments involving each type of DC recipient, with imaging conducted 4 to 6 hours after B cell transfer. The vertical lines indicate medians.

We next examined the dynamic behavior of larger populations of recently arriving B cells located within the peri-HEV region. Many MD4 cells in this setting also showed prolonged interactions with HEL-DCs, migrating slowly along short-range tracks near their DC partners (Fig. 2C and movie S7). In contrast, MD4 and non-tg B cells migrated rapidly in and out of perivascular arrays of DCs lacking specific Ags, resulting in the generation of longer-range tracks covering both DC-rich and DC-sparse areas (Fig. 2C and movie S7). Quantitative analysis 3 to 6 hours after transfer showed no measurable differences in contact time or migration speed for MD4 versus non-tg B cells in the presence of control OVA-DCs (Fig. 2D and movie S8). In contrast, MD4 cells, but not non-tg B cells, exhibited a considerable increase in contact time and reduced migration speed in the presence of HEL-DCs (Fig. 2D and movie S9).

Follicular exclusion is a well-established property of Ag receptor–engaged B cells, a phenomenon characterized by the localization of these cells to the outer T cell zone (25). Because our results revealed a series of Ag-specific B cell–DC interaction events within the T cell zone that influenced B cell mobility, we tested whether HEL-DCs would prevent or retard follicular entry by MD4 B cells 10 to 12 hours after transfer into mice that had previously received DCs. This was a time point at which many MD4 cells showed phenotypic evidence of activation in the presence of HEL-DCs (Fig. 3A). Although MD4 and non-tg B cells homed to follicles with a similar efficiency after arriving in LNs containing OVA-DCs, MD4 B cells were preferentially retained in the T cell zone in HEL-DC recipients (Fig. 3B). Consistent with this result, the expression of the chemokine receptor CCR7 by MD4 B cells was increased in this situation (fig. S6) (26).

Follicular exclusion of B cells activated by Ag-carrying DCs. (A) Ten to 12 hours after being transferred into mice injected with HEL-DCs, OVA-DCs, or phosphate-buffered saline (PBS), carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled MD4 B cells were recovered from draining inguinal LNs for fluorescence-activated cell sorting analysis. Data are gated on CD19+CFSE+ cells. One of three similar experiments is shown. (B) (Left) Distribution of co-transferred MD4 (red) and non-tg B cells (green) on sections of LNs containing OVA-DCs or HEL-DCs. LNs were taken from recipient mice 10 to 12 hours after B cell transfer. Each B cell is highlighted with a circle for clarity. ERTR-7 staining (white) is used to distinguish T cell areas from follicles, a technique established in fig. S1. Scale bar, 100 μm. (Right) The percentage of extrafollicular B cells in recipients of the indicated DC population. Data were collected from randomly selected sections or fields and are presented as mean ± SEM of three independent experiments. * P < 0.01 by t test.

A hallmark of specific B lymphocyte activation is BCR-mediated acquisition of Ags. This allows B cell presentation of the MHC molecule–associated peptides required for development of T cell–dependent antibody responses. In accord with the preceding data on activation and migration, a substantial majority of MD4 cells acquired HEL 3 to 4 hours after arriving in a LN containing HEL-DCs (fig. S7). Analysis of the HEL distribution pattern in B-DC pairs on stained LN sections revealed that non-tg B cells failed to acquire HEL from the DCs they contacted, which typically stained for HEL throughout the cell interior (Fig. 4A) and sometimes along dendrites (fig. S8). In contrast, considerably less HEL remained associated with DCs engaged by MD4 cells, and the Ag was instead concentrated in the Ag-specific B cell partner (Fig. 4, B and C). These data indicate that BCR-mediated Ag acquisition occurs during direct B-DC interactions in the T cell zone rather than as a result of the spontaneous release of Ags from the DCs, a conclusion consistent with the migration analysis detailed above (Fig. 2).

B cell acquisition of Ags from DCs. (A and B) Sections were obtained from LNs of HEL-DC recipients taken 3 hours after B cell transfer and stained for HEL. Examples of HEL-pulsed DCs in contact with non-tg (A) or MD4 B cells (B) are shown as overlays of all three colors (left), only transferred DCs and B cells (center) to highlight cell-cell contacts, and only B cells and HEL staining (right) to highlight the difference in Ag distribution. Scale bar, 5 μm. (C) 153 HEL-pulsed DCs in contact with MD4 B cells and 157 in contact with non-tg B cells were scored for amounts of HEL in randomly selected fields of four LNs for each condition collected from two independent experiments. The Mann-Whitney rank sum test was used to compare amounts of HEL associated with HEL-pulsed DCs in contact with MD4 cells versus with non-tg B cells.

The extrafollicular localization of receptor-engaged B cells is likely dictated by the need for cognate T-B interactions in the T cell zone before further development of T-dependent antibody responses (27). Here, we used intravital two-photon imaging to document that upon LN entry, B cells survey local Ag-carrying DCs. Specific B cells recognizing their Ags on these DCs are activated and arrested in the T cell zone before their follicular homing (Note S1). These results are not in conflict with the available evidence that diffusing low-molecular-weight Ags may engage specific B cells inside follicles and induce their migration toward the T cell zone (28, 29) (Note S2); rather, they point to a mechanism that, given the wide variety of Ag-capturing mechanisms possessed by DCs, may provide B cells with broader access to Ags, particularly those of large sizes or associated with particulate materials. In addition, presentation of membrane-tethered Ags would also facilitate activation of low-affinity B cells in the naïve repertoire (30). Lastly, through presentation of both T cell and B cell epitopes derived from the same Ag, DCs could serve as a cellular platform to facilitate activation, colocalization, and mutual communication of rare Ag-specific T and B cells, whose interaction ultimately leads to antibody responses.

We are grateful to O. Schwartz at the NIAID Biological Imaging Facility for expert advice on confocal microscopy; I. Ifrim and A. Rinker for research assistance; and R. Casellas, J. Cannons, and P. Schwartzberg for comments on the manuscript. H.Q. and A.Y.C.H. received Cancer Research Institute Postdoctoral Fellowships. H.Q. is particularly in debt to Y. Hong for support, encouragement, and inspiration. This work was supported by funds from the Intramural Program, NIAID, NIH, U.S. Department of Health and Human Services.